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Purification method for lactoferrin and lactoperoxidase

A lactoperoxidase and purification method technology, which is applied in the field of lactoferrin and lactoperoxidase purification, can solve the problems of cumbersome methods, small processing capacity, and difficulty in large-scale production, and achieve the effect of low cost

Active Publication Date: 2014-04-09
北京济普霖生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hydroxyapatite chromatography is composed of hexagonal HAp ceramics, which can strongly bind natural proteins. Once the protein denatures, this binding force decreases rapidly. Therefore, the degree of protein retention on this support medium is not easy to predict.
[0006] There are many patents on the method of separating and purifying lactoferrin from milk and lactoperoxidase and the patents on the antibacterial activity of lactoferrin. For example, the invention patent of Chinese patent application number 200710102035.6 discloses a The method removes casein to obtain clarified whey, and then uses SP Sepharose HP chromatographic column to separate and purify recombinant human lactoferrin. It can be seen that the chromatographic filler used in this method has a small particle size and will generate high pressure, so Limits the range of flow rates
The invention patent with the Chinese patent application number 200610040528.7 discloses the use of bovine colostrum defatted whey as raw material, the use of enzymatic method to remove casein, the combination of membrane separation technology and industrial chromatography technology, and the separation purity from bovine colostrum is more than 90%. lactoferrin, but this method is cumbersome and costly
[0007] It can be seen that some separation and purification technologies can prepare high-purity lactoferrin on a laboratory scale, but the processing volume is small, the cost is high, and it is difficult to produce on a large scale

Method used

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  • Purification method for lactoferrin and lactoperoxidase
  • Purification method for lactoferrin and lactoperoxidase
  • Purification method for lactoferrin and lactoperoxidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 contains the pretreatment of recombinant human lactoferrin milk sample

[0029] Collection of milk samples from mammals containing recombinant human lactoferrin (0.1-5 g / L), including milk samples from transgenic animals containing recombinant human lactoferrin, or non-transgenic milk mixed with recombinant human lactoferrin Animal milk samples, where milk samples are preferably fresh liquid milk samples, or liquid milk samples obtained after thawing frozen samples or reconstituted milk powder. Mammalian milk samples include cow milk, goat milk, rabbit milk, etc., with milk as the preferred. First, the milk containing recombinant human lactoferrin is skimmed through a butterfly centrifuge to obtain skim milk with a fat content of less than 0.2%, and then 1.4 μm ceramic membrane technology is used to remove bacteria in the milk. The operating temperature is 0-35 ° C. The inlet pressure is 0.3MPa~0.5MPa, then open the permeation valve, set the operating pres...

Embodiment 2

[0030] Embodiment 2 SP Sepharose Big Beads cation exchange chromatography separation

[0031] The skim milk prepared in Example 1 was loaded onto the SP Sepharose Big Beads cation exchange chromatography column equilibrated with 5mM EDTA, 3ms / cm, pH6.5 phosphate buffer, and the flow rate was 150cm / h. After loading, use Wash the chromatography column with 5mM EDTA, 3ms / cm, pH6.5 phosphate buffer to the baseline, use 1%Triton X-114, 13ms / cm NaCl, 3ms / cm phosphate buffer (pH6.5) to The protein solution of lactoperoxidase was eluted and analyzed for purity by gel filtration chromatography, and the protein purity was ≥80%. Since lactoferrin can bind tightly with endotoxin, the antibacterial ability of lactoferrin is limited, and the use of non-ionic detergent can encapsulate endotoxin in the capsule and metal complexing agent can form a gap between endotoxin and protein. The calcium bridge of the recombinant human lactoferrin is broken, so the present invention adopts the method o...

Embodiment 3

[0032] Embodiment 3SP Sepharose Fast flow cation exchange chromatography separation

[0033] Pack the commercially available SP Sepharose Fast flow filler into the XK26 / 40 chromatographic column. The skim milk prepared in Example 1 was loaded onto the SP Sepharose Fast flow cation-exchange chromatography column equilibrated with 4ms / cm pH6.0 phosphate buffer, the flow rate was 74cm / h, and after the loading was completed, use 4ms / cm cm, pH 6.0 phosphate buffer solution to wash the chromatography column until the 280nm absorption signal reaches the baseline, then use 11ms / cm NaCl, 4ms / cm phosphate buffer solution (pH 6.0) to wash the protein solution containing lactoperoxidase Take it off, and then use 84ms / cm NaCl, 4ms / cm phosphate buffer (pH6.0) to single-elute lactoferrin, and perform purity analysis by gel filtration chromatography. The protein purity is ≥95%. At this time, the protein is pure The product solution contains 5% bovine endogenous lactoferrin. figure 2 It is ...

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Abstract

The invention provides a purification method for recombination human lactoferrin, animal lactoferrin and lactoperoxidase. The method comprises: taking a milk sample containing recombination human lactoferrin as a raw material, pre-treating to obtain degreased milk, performing cation exchange chromatographic separation on the obtained degreased milk, then under different salt concentration conditions, performing elution separation on lactoferrin and lactoperoxidase, so as to obtain high-purity lactoferrin and lactoperoxidase, and further performing separation on the solution rich in lactoferrin by utilizing hydrophobic interaction chromatography to respectively obtain recombination human lactoferrin and animal source lactoferrin both with the purity of 99% or more. The purity of recombination human lactoferrin obtained by utilizing the purification method provided by the invention is 99% or more, and also recombination human lactoferrin has the physical and chemical activities and physiological functions all similar to those of natural human lactoferrin; and the technology is a one-step purification process, so that the purification method is low in cost, simple and suitable for industrialized large-scale production.

Description

technical field [0001] The invention relates to protein purification, in particular to a method for purifying lactoferrin and lactoperoxidase. Background technique [0002] Lactoferrin (Lactoferrin, LF) is a non-heme iron-binding protein and a member of the transferrin family. It, together with serum transferrin and ovotransferrin, has the function of transferring iron to serum. In addition, lactoferrin also has the following important physiological functions: (1) Participate in iron metabolism in the body, promote iron absorption and utilization, improve the effect of iron biopharmaceuticals and reduce their side effects, and prevent and treat anemia caused by iron deficiency (2) Broad-spectrum antibacterial activity and antiviral activity; (3) Improve gastrointestinal function and promote infant gastrointestinal development; (4) Anti-inflammatory activity and immune regulation function; (5) Antioxidant Function: Lactoferrin can prevent the formation of active oxygen free ...

Claims

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Application Information

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IPC IPC(8): C07K14/79C07K1/18C07K1/20C12N9/08
CPCC07K14/79C12N9/0065C12Y111/01
Inventor 赵建敏王建武王慧敏李由付明波汤波李宁
Owner 北京济普霖生物技术有限公司
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